Inhibitor Of Acetylcholine Transport Research Articles

Evidence to suggest that extracellular acetate is accumulated by rat hippocampal cholinergic nerve terminals for acetylcholine formation and release

It is well established that extracellular choline is transported into central cholinergic nerve terminals by `high' and `low' affinity processes to form the neurotransmitter acetylcholine (ACh). The intent of the present investigation was to ascertain whether extracellular acetate might also be transported into central cholinergic nerve terminals to form ACh. To test this possibility, rat hippocampal tissue was incubated with varying concentrations of extracellular [1- 14C]acetate (0.1–100 μM) and the uptake of [1- 14C]acetate and the amount of [ 14C]ACh formed by the tissue determined. The results indicated that the uptake of extracellular [1- 14C]acetate was temperature-dependent and saturable having an apparent Michaelis constant ( K m) of 22 μM. The formation of [ 14C]ACh in the tissue as a function of extracellular [1- 14C]acetate appeared to occur by both `high' and `low' affinity processes with apparent K m values of 0.5 and 19.6 μM, respectively. In other experiments, three inhibitors (lithium, allicin and sodium) of acetyl CoA synthetase (EC 6.2.1.1 acetate: CoA ligase), the enzyme which converts acetate to acetyl CoA when ATP and CoA are present, inhibited [1- 14C]acetate uptake and the amount of [ 14C]ACh formed from that [1- 14C]acetate. Additionally, vesamicol, an inhibitor of ACh transport into synaptic vesicles, blocked the filling of a synaptic vesicle-enriched fraction of hippocampal tissue with newly synthesized [ 14C]ACh formed from extracellular [1- 14C]acetate. High K + depolarization of hippocampal tissue loaded with extracellular [1- 14C]acetate not only increased the synthesis but also the release of [ 14C]ACh. These results suggest that extracellular acetate is recycled by rat hippocampal cholinergic nerve terminals for the formation and release of ACh. They also suggest that the enzyme acetyl CoA synthetase mediates extracellular acetate uptake into hippocampal cholinergic nerve terminals by metabolizing it to acetyl CoA and thereby creating a diffusion gradient for it to follow. © 1997 Elsevier Science B.V. All rights reserved.

Monoethylcholine as a flase transmitter precursor at the fog and mouse neuromuscullar junctions

Monoethylcholine (MECH0 enters motor nerve terminals where it is made into acetylmonoethylcholine (AMECH). AMECH opens endpate channels for about half of the average duration observed when they are opened by acetylcholine (ACH). Therefore when AMECH is present in a quatum the endplate currents decay more rapidly. MECH has been used to measure quantal turnover in motor nerve terminals. We find that the incorporation of AMECH into quanta is blocked by vesamicol, and inhibitor of ACH transport into synaptic vesicles. AMECH is incorporated more rapidly when acetylcholinesterase is inhibited, when the choline uptake inhitor, hemicholinium-3, is present or when extracellular Na + (required for active CH uptake) is replaced with methylamine. This suggests that in the absence of these inhibitors CH obtained from released ACH is recycled. Therefore, experiments on the rate of incorporation of MECH are misleading unless CH recycling is prevented. Previous work also suggested that MECH is incorporated at a faster rate into those quanta which are released by stimulation than into those released spontaneously. We concluded that quanta released spontaneously and following nerve stimulation proabably come from the same pool. The distribution of t 1 2 's during the incorporation of MECH can be accounted for in the framework of recent studies of the recyclingt of synaptic vesicles. We concluded that false transmitter is a valuable tool for studying the loading of quanta, but that there are several complications to be considered when trying to use it to measure the turnover of the population of quanta.

Acetylcholine synthesis is modulated by acetylcholine content of cytosolic fraction but not by that of releasable fraction

Synthesis and release of acetylcholine (ACh) in the rat hippocampal slices were examined to clarify the mechanism of modulation of ACh synthesis. Treatment with 2-(4-phenylpiperidino)cyclohexanol (AH5183, 50 μM), an inhibitor of ACh transport from cytosol to synaptic vesicles, inhibited the increase in ACh content of the membrane-bound fraction which is readily releasable, but did not affect the cytosolic ACh content. Under these conditions, the total ACh content reached a plateau value. These results indicate that ACh synthesis is modulated by cytosolic ACh content but not by the vesicular fraction.

Effect of N, N′-dicyclohexylcarbodiimide on the binding of vesamicol, an inhibitor of acetylcholine transport into synaptic vesicles

Vesamicol is a highly potent inhibitor of active acetylcholine transport into isolated cholinergic vesicles from Torpedo. On the basis of transport kinetics and vesamicol sensitivity, we have shown that the acetylcholine transporter could be in an activated state even in the absence of a stimulated ATPase. In this preparation, N, N′-dicyclohexylcarbodiimide (DCCD), an hydrophobic carbodiimide, inactivates both ACh transport and vesamicol binding. Inhibition of vesamicol binding by DCCD is time dependent, saturable and prevented by vesamicol. DCCD first affected the affinity constant for vesamicol. K i-value for DCCD lies in the micromolar range. These results imply that there is a DCCD reactive site within the ACh transporter and that it is located in an hydrophobic environment near the vesamicol binding site. SDS gel electrophoresis after labelling of the vesicle membrane proteins with [ 14C]DCCD shows that radioactivity is mainly incorporated in a 15 kDa subunit. Time-course and concentration dependence of [ 14C]DCCD labelling and vesamicol inhibition do not coincide. Hence, the two processes are probably unrelated and the result rather points to another inactivation mechanism which can be an intramolecluar cross link.

Acetylcholine recycling and release at rat motor nerve terminals studied using (-)-vesamicol and troxpyrrolium.

1. The presynaptic mechanisms governing the release and recycling of synaptic vesicles have been studied by examining the effects of nerve stimulation, (-)-vesamicol (an inhibitor of acetylcholine transport into synaptic vesicles) and troxypyrrolium (an inhibitor of the high-affinity, sodium-dependent, choline uptake system) on endplate currents (EPCs) and miniature endplate currents (MECPs) recorded from motor endplates in cut rat hemidiaphragm preparations. 2. In control experiments, 5 min of 10 Hz nerve stimulation had no effect on either the mean or the distribution of MEPC amplitudes. 3. Nerve stimulation in the presence of (-)-vesamicol (25 nM-10 microM) revealed a population of MEPCs that was unaffected by the compound and a population of MEPCs whose mean amplitude was selectively reduced by the compound. 4. Nerve stimulation in the presence of troxypyrrolium (20 microM) produced a uniform reduction in the amplitude of all MEPCs with no change in the coefficient of variance of MEPC amplitudes. 5. The concentration-dependent effects of (-)-vesamicol on the amplitude of the evoked EPCs paralleled the concentration-dependent effects of the compound on MEPC amplitudes. 6. The results are consistent with the hypothesis that both recycled and performed synaptic vesicles are heterogeneously released from rat motor nerve terminals and that (-)-vesamicol acts selectively on recycling vesicles. In addition, a model of vascular loading that accounts for the different effects of nerve stimulation on MEPC amplitudes in the presence of (-)-vesamicol and troxypyrrolium is described.